MiRNA-185 Regulates The VEGFA Signaling Pathway In Dairy Cows With Retained Fetal Membranes

Retention of fetal membranes(RFM)is an common reproductive disturbance in dairy cows,and is related to miRNAs by our team.mi RNA-185 as one of differential expressed miRNAs is significantly highly expressed which has a larger fold-change than other all miRNAs.Vascular endothelial growth factor(VEGF)A,regulated by miRNA-185 in human breast carcinogenesis test,can activate arachidonic acid(ARA)release via the VEGFA signaling pathway,which influences RFM.These suggest that miRNA-185 plays an important role in RFM of cows.The aim of this study was to explore the pathogenic mechanism of RFM by investigating the regulatory relationship between miRNA-185 and the VEGFA signaling pathway.The paper can be summarized as the following three parts: Caruncle tissues were collected from healthy and RFM cows to detect VEGFA m RNA and protein levels using Q-PCR and WB.And the expression level and position of VEGFA mRNA were detected by FISH technology.Uterine caruncle epithelial(UCE)cells were cultured by the explant culture method,further purified,and to verified the target relationship of mi RNA-185 and VEGFA by dual-luciferase reporter gene assay.Subsequently treated with VEGFA inhibitor,MEK inhibitor,miRNA-185 mimics and miRNA inhibitor or left untreated as a control for detection of the mRNA and protein levels,respectively,of genes involved in the VEGFA signaling pathway(VEGFA,PLC,PRK,RAF,MEK,MAPK,P-p44/42 MAPK,PLA).The cellular supernatant was collected for measurement of ARA concentrations at 12,24 and 48 h after treatment.Serum samples of healthy Holstein dairy cows and RFM cows were collected to detect VEGFA and ARA concentrations by ELISA.And Q-PCR and WB were performed to detect caruncle tissues the mRNA and proteins levels,respectively,of genes involved in the VEGFA signaling pathway.The Q-PCR and WB results showed that the mRNA and protein of VEGFA were down-regulated in the caruncle tissue of RFM cows(p < 0.01).And the FISH result showed that it was expressed in UCE cells of caruncle.The UCE cells were successfully isolated and cultured by the explant culture method,further purified,and it's purity exceed 90% after identifying with the antibody of CK18.The dual-luciferase reporter gene assay result showed that the fluorescence intensity was decreased when transfect 3'UTR-WT and miRNA-185 into the cell.In UCE cells treated with the VEGFA inhibitor,expression of VEGFA,RAF,MAPK(p < 0.05)and PLC,PRK,MEK,P-p44/42 MAPK and PLA(p < 0.01)was significantly down-regulated,meanwhile the expression of RAF was down-regulated but no significantly(p > 0.05).In UCE cells treated with the MEK inhibitor,expression of MEK,MAPK,P-p44/42 MAPK and PLA was significantly down-regulated(p < 0.01),while that of VEGFA,PLC,PRK and RAF was no significantly change(p > 0.05).In UCE cells treated with the miRNA-185 mimics,expression of VEGFA,PLC,RAF,MEK,MAPK and PLA was significantly down-regulated(p < 0.01),while that of P-p44/42 MAPK was significantly up-regulated(p < 0.01).In UCE cells treated with the miRNA-185 inhibitor,expression of VEGFA,PRK,MEK,MAPK,PLA(p < 0.01)and PLC,RAF(p < 0.05)was significantly up-regulated(p < 0.01),while that of P-p44/42 MAPK was down-regulated but no significantly change(p > 0.05).After treatment 24 and 48 h,the release of ARA was significantly increased in miRNA-185 inhibitor group(p < 0.01),as compared to other groups.Serum levels of VEGFA and ARA from RFM cows were abnormally increased at 12 h after calving,as compared to those in healthy dairy cows.Expression levels of most of the investigated genes(VEGFA,PLC,PRK,RAF,MEK,MAPK,and PLA)were down-regulated in the caruncle tissue of RFM cows.However,P-p44/42 MAPK was up-regulated in the caruncle tissues of cows with RFM(p < 0.01).These results demonstrate that expression level of VEGFA is regulated by miRNA-185,and mi RNA-185 can regulate the ARA concentration by VEGFA signaling pathway,which influences the release of the fetal placenta after calving.